Antithrombin III, or heparin cofactor, is the major plasma inhibitor of the activated blood clotting proteases. Alone, antithrombin III reacts slowly with these enzymes to form a 1:1 inactive complex. Addition of heparin greatly accelerates the rate of protease inactivation without altering the stocichiometry of the reaction. This increase in reactivitiy appears to result from heparin-induced conformational changes in antithrombin and the coagulation proteases. We have shown that site-specific chemical modificaton of antithrombin blocks heparin binding but does not interfere with the formation of the protease inhibitor complex. The interaction of antithrombin with these proteases will be studied through the use of chromophoric "reporter groups." Changes in protein conformation monitored with these chromophores will be correlated with the kinetics of protease inactivation.